Double-stranded RNA (dsRNA)-mediated gene silencing or RNA interference (RNAi) was discovered and used as a genetic tool to “knockout” gene expression in the nematode Caenorhabditis elegans (P. Sharp, Genes & Development 13:139-141, 1999). This gene silencing phenomenon was later found to be highly conserved in many eukaryotic cells. Introduction of long dsRNA into the cells of organisms leads to the sequence-specific degradation of homologous gene transcripts. The long dsRNA molecules are metabolized to small (e.g., 21-23 nucleotide (nt)) interfering RNAs (siRNAs) by the action of an endogenous ribonuclease known as Dicer (Grishok et al., Science 287:2494-2497, 2000; and Zamore et al., Cell 101:25-33, 2000). The siRNA molecules bind to a protein complex, termed RNA-induced silencing complex (RISC), which contains a helicase activity and an endonuclease activity. The helicase activity unwinds the two strands of RNA molecules, allowing the antisense strand to bind to the targeted RNA molecule (Zamore et al., Cell 101:25-33, 2000; Zamore, P. D., Science 296:1265-1269, 2002; and Vickers et al., J Biol Chem. 2003 Feb. 28; 278(9):7108-18). The endonuclease activity hydrolyzes the target RNA at the site where the antisense strand is bound. Therefore, RNAi is an antisense mechanism of action, as a single stranded (ssRNA) RNA molecule binds to the target RNA molecule by Watson-Crick base pairing rules and recruits a ribonuclease that degrades the target RNA.
Another post-transcriptional gene silencing process is mediated by micro RNA, or miRNA, a ssRNA species which suppress mRNA translation (Lee et al., Cell 75, 843-54 (1993)). Like siRNA, miRNA are derived from RNA precursors that are processed to 21-25 nt sequences by endonuclease Dicer and form a sequence specific gene silencing complex. See, McManus & Sharp, Nat Rev Genet 3, 737-47. (2002).
In mammalian cells, dsRNA longer than 30 bp can cause non-specific gene suppression by an interferon α response. However, cells transfected with 21 nt synthetic double-stranded siRNA bearing two nucleotides protruding at both 3′-ends may escape an interferon response and effectively exert sequence-specific gene silencing function. The silencing effect of the synthetic siRNA, however, is transient. Plasmid DNA expressing siRNA has also been developed utilizing transcription systems including T7 polymerase, and mammalian pol II or pol III promoters. Wang et al., J Biol Chem 275, 40174-9 (2000); Yu et al., Proc Natl Acad Sci USA 99, 6047-52 (2002). The effectiveness of gene silencing by siRNA-encoding plasmids depends on DNA transfection efficiency, which can be low for many cell types and, in particular, for in vivo studies. Plasmid DNA transfection also results in transient siRNA expression. For effective gene silencing, e.g., as might be desired for a gene therapy application, a system that provides high levels of siRNA expression for prolonged periods would be desirable.